Four new yeast strains are obtained by: (1) fusion between Saccharomyces cerevisiae (Montrachet) and Schizosaccharomyces pombe; (2) by fusion between the yeast strain formed in (1) above and the parental strain of Saccharomyces cerevisiae; (3) by fusion between the yeast strain formed in (1) above and a yeast with the Killer phenotype; and (4) by fusion between the product formed in (1) above a Saccharomyces cerevisiae denominated as "O Mendoza".
These new yeasts were respectively designated (1) MB7TC.alpha.; (2) MBII; (3) MBK; and (4) MBO.
The invention also concerns a process for the reduction of malic acidity in wine making.
A wine is considered acidic when it contains an elevated concentration of L-malic acid, which in turn, is produced by fermentation of musts from not fully ripened grapes. L-malic acid is one of the acids present in musts and wines, together with tartaric acid, citric acid, and much smaller amounts of oxalic acid, uronic acid, succinic acid, lactic acid, acetic acid, glioxalic acid, glyceric acid, pyruvic acid, oxaloacetic acid, alpha-ketoglutaric acid, etc.
The two major acids in terms of concentration, tartaric and malic acids, are metabolized to sugar during the process of grape maturation on the vine. At the end of maturation, malic acid undergoes the greatest rate of degradation, with concentrations being reduced from 200 mEq/l to less than 50 mEq/l, while tartaric acid is degraded from 150-200 mEq/l to 100-150 mEq/l. The metabolization of tartaric acid leads to its lowest concentrations almost one month before the end of grape maturation. Malic acid degradation continues to increase up until full grape maturation. As mentioned earlier, acid musts result from the harvesting of grapes that did not go through the full maturation process. Thus, when fermented, these musts also result in acid wines.
Since tartaric acid present in musts is stable to the action of bacteria and yeasts, the correction of acidity in wines by biological processes is limited to malic acid degradation. Microbial degradation of malic acid can occur through the action of bacteria of the genera Pediococcus, Lactobacillus and Leuconostoc via malolactic fermentation, with the formation of lactic acid, or by degradation to ethanol via the action of yeasts that develop maloalcoholic fermentation.
Malolactic fermentation primarily occurs in wines with acidity of less than 1 g/l tartaric acid and pH higher than 3.1. Fermentation with homofermenting bacteria of highest interest for wine should occur at pH between 3.2 and 3.3 (MENDOZA, 1979).
Schizosaccharomyces pombe yeasts have been used to decrease malic acid in wine by CASTELLI (1969), CASTELLI and HAZNEDARI (1971), BENDA and SCHIMITT (1966), BENDA (1974) HUGLIN et al. (1976), GALANDER (1977) and CARRAU (1981). However, owing to problems related to the growth rate of Schizosaccharomyces pombe which require the use of large inocula for wine fermentation (CARRAU, 1982), the use of this yeast for demalification is not practical for common application in wineries. Indeed, one of the objectives of the present invention was to construct yeasts by fusing spheroplasts of Saccharomyces cerevisiae (having a greater growth rate) and Schizosaccharomyces pombe. These yeasts can solve the problems outlined above because their growth rate is more rapid than that of the parental strain Schizosaccharomyces pombe and because they express the ability to degrade L-malic acid in addition to presenting part of the genome of yeasts traditionally used in wine fermentation.
A decrease in malic acid content also favors the natural development of malolactic fermentation which is known to be a determinant of the biological stabilization of wines.
The present invention was based on the use of microbial genetics, to whose methods protoplast fusion was added during the 1970's. This method permits the exchange of genetic material between cells that are not compatible for crossing. For yeasts, protoplast fusion utilizes lytic enzymes from the digestive juice of Helix aspersa which produce the formation of cells fully (protoplasts) or partially (spheroplasts) devoid of walls. Both protoplasts and spheroplasts can be easily fused in the presence of polyethylene glycol, as initially observed by VAN SOLINGEN and VAN DER PLATT (1977). The fusion products are then submitted to conditions that will lead to the reestablishment of the cell wall. These conditions include a regenerating medium containing Ca.sup.++ and a large osmotic concentration, with inoculation onto pour-plates yielding the best percentage of regeneration. This technique has been used previously to obtain fusion products of technological interest (RUSSEL and STEWART, 1979; TUBB, 1979). Modern wine making outfits use selected yeasts that are applied to the fermentation by the use of starter. Carrau (1982) reported the demalifying ability of six different strains of Schizosaccharomyces pombe, which degraded 55 to 75% of malic acid within twenty-four hours and 83.8% to 98.8% after seventy-two hours. On the basis of this ability of Schizosaccharomyces pombe to degrade malic acid, (CARRAU, 1979) proposed large-scale fermentations using this yeast to degrade malic acid in wine. This process would utilize inocula of 500.times.10.sup.5 cells of Schizosaccharomyces pombe in order to obtain partial malic acid degradation during wine fermentation.